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Arichard:Protocols/MDA amplicon extraction and processing
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====Amplicon processing==== # Briefly spin samples to collect fluid. # Per reaction, add to UV treated 10X Phi29 buffer and H2O: ## 1 ul 10 mM dNTP ## 1 ul 370 ng/ul N6 ## 0.5 ul Phi29 # Add 1.5 ul ALS, incubate 3 minutes at RT. Mix well by pipette. # Move reaction to ice block and add 1.5 ul NS. Mix well by pipette. # Add 16 ul DNA Pol I master mix to each tube. Mix well by pipette and incubate 30 minutes at 37 degC, followed by 10 minutes at 65 degC. # Add reaction to ethanol precipitation mix. Incubate 20 minutes at -80 degC. # Spin 25 minutes at 10,000 RPM (10,600 RCF) at 4 degC. # Carefully remove and discard supernatant and add 750 ul of 75% EtOH to each pellet. # Spin 5 minutes at 10,000 RPM at 4 degC. # Carefully remove and discard supernatant and let dry 5 minutes. # Add 7 ul H2O to each pellet and resuspend by pipetting. Store at 4 degC or proceed directly to tagmentation and amplification.
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